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Frontiers in Food Science and Technology ·

Volume 6 - 2026 | https://doi.org/10.3389/frfst.2026.1927350

Soybean variety influences the quality and functionality of soymilk fermented by Lactobacillus acidophilus 1132

  • Muzaffar Hasan1,2*corresponding author
  • Dilshad Ahmad3,4
  • Veda Krishnan1
  • Archana Sachdev1
  • Anil Dahuja1*corresponding author
  1. 1Division of Biochemistry, ICAR-Indian Agricultural Research Institute, New Delhi, India
  2. 2Centre of Excellence on Soybean Processing and Utilization, ICAR-Central Institute of Agricultural Engineering, Bhopal, India
  3. 3Division of Seed Science and Technology, ICAR-Indian Agricultural Research Institute, New Delhi, India
  4. 4ICAR-National Institute of Seed Science & Technology, Mau, Uttar Pradesh, India
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Abstract

Fermented soymilk is a widely consumed plant-based beverage, and fermentation enhances its nutritional and functional properties. However, the influence of soybean seed characteristics on the quality and functionality of fermented soymilk remains insufficiently understood. Therefore, this study investigated the effect of three soybean varieties-food-grade (Karune), low-KTI (V23), and black (VL-Bhat-65)- on the nutritional, functional, technological, and sensory properties of soymilk fermented with Lactobacillus acidophilus 1132. All soybean varieties supported bacterial growth and lactic acid production. Fermentation significantly (p < 0.05) improved the nutritional and functional quality of soymilk by reducing raffinose-family oligosaccharides (RFOs) and antinutritional factors (ANFs), while enhancing isoflavone bioconversion and phenolic content. Black soybean soymilk exhibited the highest protein and mineral contents and viscosity; food-grade soymilk showed the greatest antioxidant activity, lowest phytic acid and trypsin inhibitor activity, and highest sensory acceptability, whereas low-KTI soymilk demonstrated the greatest conversion of glucoside to aglycone isoflavones. These findings demonstrate that soybean variety significantly influences the quality and functionality of fermented soymilk and provide a scientific basis for selecting appropriate soybean varieties for the development of nutritionally enhanced fermented plant-based beverages.

Keywords

1 Introduction

Soymilk has received increasing attention from consumers as an alternative to dairy products due to its high protein quality, absence of cholesterol and lactose, and abundance of bioactive compounds, particularly isoflavones (Battistini et al., 2018; Hasan et al., 2023; Hasan et al., 2022). It is also appreciated as an affordable substitute for cow’s milk for the human population suffering from lactose intolerance, milk protein allergy, and those who have eliminated milk from their diet due to other reasons (O’Reilly et al., 2001; Xu and Chang 2009a; Hasan et al., 2022). Owing to these attributes, soy-based foods have been shown to provide positive effects in alleviating menopausal symptoms, reducing the risk of CVDs (cardiovascular disease), bone health, and lowering the likelihood of breast cancer in females and prostate cancer in males. These health benefits are attributed to their high concentration of bioactive compounds such as phenolic compounds (as isoflavones) (Devi et al., 2009; Hasan et al., 2023). Soymilk contains twelve major isoflavones, including the aglycones genistein, daidzein, and glycitein, together with their glucoside, acetyl-, and malonyl-derivatives, which contribute substantially to its nutritional and functional properties (Wang and Murphy, 1994; Prabhakaran et al., 2006).

In addition to the nutritional components, other antinutritional factors are also present in the soymilk. These antinutritional factors can be removed or overcome by different processing techniques, such as germination, fermentation, and other methods (Murugkar, 2015). In addition to overcoming these ANFs, processing techniques help improve the nutritional and functional properties of soymilk. Fermentation of the soymilk by the probiotic bacteria also improves the flavor and texture of the fermented soymilk (Sasi et al., 2023).

One of the most widely used and studied probiotic strains is Lactobacillus acidophilus, a homofermentative LAB strain (María Remes-Troche et al., 2020). There is evidence that it has therapeutic effects on the human body, such as lowering cholesterol, enhancing immunity, preventing diarrhea, and inhibiting several bacterial pathogens (Gao et al., 2023). Several physicochemical properties of soymilk, including its pH, viscosity, and nutrient composition, play a crucial role in its nutritional quality and sensory attributes (Olías et al., 2023). Soymilk generally has a pH ranging from neutral to mildly acidic, creamy consistency. These properties can be significantly altered by fermentation (Harper et al., 2022; Statsenko et al., 2023). Fermentation with Lactobacillus acidophilus can boost the nutritional profile of soymilk by enhancing the bioavailability of isoflavones and other nutrients (Kumari et al., 2022; Riasatian et al., 2023). During fermentation, the bacteria produce lactic acid, an acid that decreases the pH of soymilk and can lead to the degradation of anti-nutritional factors, thereby enhancing nutrient absorption (Arbab Sakandar et al., 2023). Fermentation significantly influences the physical properties of soymilk in various ways. The decrease in pH during fermentation can lead to protein coagulation, altering the smoothness and thickness of the final product. These transformations play a vital role in shaping the sensory attributes of fermented soymilk, including its taste, texture, and aroma, all of which are crucial for consumer appeal. Additionally, fermentation can produce bioactive peptides with antioxidant and anticancer properties, further enhancing their health benefits (Siddiqui et al., 2024). In this study, L. acidophilus 1132 was selected because strains of this species exhibit β-glucosidase activity, enabling the conversion of glucoside isoflavones into more bioavailable aglycone forms, while simultaneously producing lactic acid that contributes to product acidification and microbial stability.

Soybean varieties differ considerably in seed coat pigmentation and biochemical composition, including protein quality, phenolic compounds, mineral composition, and antinutritional factors. Black soybean (VL-Bhat-65) is characterized by anthocyanin-rich seed coats and elevated phenolic content, whereas the low-KTI variety (V23) possesses genetically reduced Kunitz trypsin inhibitor activity, and the food-grade cultivar (Karune) has desirable processing characteristics. These compositional differences are expected to influence microbial fermentation, isoflavone bioconversion, and the nutritional and sensory quality of fermented soymilk. The compositional differences in the varieties can also be an influencing factor in affecting the nutritional and functional properties of the soymilk. Previous studies have mentioned that the processing operations and conditions employed during soymilk production significantly affect the final content and profile of bioactive polyphenols (Huang et al., 2006). Researchers have also reported that the polyphenolic profile of final soymilk is directly influenced by soybean cultivar, time of soaking, ratio of soybean to water, filtration, thermal treatment, and components influencing β-glucosidase activity (Ismail and Hayes, 2005; Jung et al., 2008).

Although several studies have reported the beneficial effects of lactic acid fermentation on soymilk quality, most have focused on a single soybean cultivar or specific quality attributes. Comparative information on how soybean varieties differing in seed composition influence nutritional, functional, technological, sensory, and isoflavone characteristics under identical fermentation conditions remains limited. Consequently, the contribution of soybean variety to the overall quality of fermented soymilk has not been systematically established. Therefore, the objective of this study was to evaluate the influence of three soybean varieties differing in seed characteristics-food-grade (Karune), low-KTI (V23), and black (VL-Bhat-65)- on the nutritional, functional, technological, sensory, and isoflavone characteristics of soymilk fermented with L. acidophilus 1132 under identical processing conditions. We hypothesized that compositional differences among the soybean varieties would significantly influence fermentation behaviour and the quality of the resulting fermented soymilk.

2 Materials and methods

2.1 Chemicals and bacterial strains

All chemicals required for nutritional and functional analysis were sourced from Sigma-Aldrich, USA. HPLC-grade acetonitrile and water were obtained from MERCK, United States. Isoflavone standards, including diadzin, diadzein, genistin, genistein, glycitin, and glycitien, as well as standards for raffinose, stachyose, and sucrose, were also purchased from Sigma-Aldrich, United States.

Lactobacillus acidophilus 1132 was obtained from the Division of Microbiology, ICAR-Indian Agricultural Research Institute, New Delhi, India. The culture was maintained in MRS broth at 4 °C and propagated at 37 °C to the late exponential phase before inoculum preparation.

2.2 Soymilk preparation

Three soybean varieties one is variety Karune (pale yellow color), the second is V23 (yellow color), and the third is VL-Bhat-65 (Black color) were used for the soymilk preparation (Figure 1). The food-grade soybean (Karune) is a cultivar developed for soy food applications with desirable processing quality; the low-KTI soybean (V23) possesses genetically reduced Kunitz trypsin inhibitor activity; and the black soybean (VL-Bhat-65) is characterized by anthocyanin-rich seed coats and enhanced phenolic content (Hasan et al., 2024; Hasan et al., 2023; Nair et al., 2023). Soymilk was prepared from these varieties using the hot-water extraction method. Soybean seeds were cleaned and soaked in distilled water overnight. The swollen soybean seeds were manually dehulled and ground with hot water (80 °C) in a 1:10 ratio, and filtered through a double layer of cheesecloth to remove insoluble residues (Donkor et al., 2007). The extracted soymilk was poured into a glass bottle, sterilized by autoclaving at 121 °C for 15 min, cooled to room temperature, and stored at 4 °C for 48 h until further use. These abbreviations are used for the prepared soymilk samples: FGSM for food-grade soymilk, LKSM for low KTI soymilk, and BSM for black soymilk.

Different soybean varieties are used for the soymilk preparation.
Figure 1. Different soybean varieties are used for the soymilk preparation.

2.3 Fermentation of soymilk

The soymilk samples were separately inoculated with 1% (v/v) of an actively growing L. acidophilus 1132 culture (1 mL inoculum per 100 mL sterile soymilk). The bacterial culture was propagated in MRS broth to the late exponential phase, and the inoculum was standardized to an initial cell concentration of 6.19 log CFU/mL, with viability confirmed by plate counting before inoculation. Fermentation was carried out in sterile 250 mL Erlenmeyer flasks containing 100 mL of soymilk under static conditions at 37 °C for 48 h, which was selected based on the optimum growth conditions of L. acidophilus 1132 and previous studies demonstrating efficient acidification and β-glucosidase-mediated isoflavone bioconversion. Uninoculated sterile soymilk incubated under identical conditions served as the control. Following fermentation, the samples were immediately cooled to 4 °C to terminate fermentation and stored under refrigerated conditions until further analyses.

2.4 Chemical analysis of fermented soymilk

The chemical composition of the soymilk samples after 48 h of fermentation was analyzed using AOAC 2000 methods. Moisture content was determined (method no. 968.11), along with total fat (AOAC 2000), crude proteins (using a conversion factor of 5.71, method no. 991.20), ash (method no. 945.46), dietary fiber (enzyme-gravimetric method: 985.29), and total carbohydrates (calculated by the differential method). Caloric values were estimated using Atwater conversion factors (proteins × 4, lipids × 9, soluble dietary fiber × 2, and carbohydrates × 4). pH levels were measured using a digital pH meter, and total titratable acidity (TTA) was assessed through titration with 0.1 M NaOH, expressed as a percentage.

To quantify minerals such as iron (Fe), zinc (Zn), potassium (K), and calcium (Ca), the samples were first dried in an oven at 80 °C. Subsequently, 1 g of the dried samples was digested in a mixture of HNO3 and HClO4. The concentrations of Fe, Zn, and Ca were measured using a Fast Sequential atomic absorption spectrometer (Agilent 240FS AA), while potassium (K) content was determined using a photoelectric flame photometer.

2.5 Microbial analysis of fermented soymilk

The viability of probiotic cells was assessed using the plate dilution technique with MRS agar (de Man et al., 1960). Fermented soymilk samples were serially diluted and plated in duplicate, followed by incubation at 37 °C for 48 h. The results were reported as colony-forming units per millilitre (cfu/mL).

2.6 Functional analysis of fermented soymilk

2.6.1 Determination of antioxidant activity

With slight modifications to the method described by Morales-de la Peña et al. (2010), the inhibition of the 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radical was assessed to determine the anti-radical activity of soymilk. A 0.1 g sample of dry soymilk powder was mixed with 1 mL of NaOH and shaken for 2 h. The mixture was then centrifuged at 10,000 rpm for 10 min, and the supernatant was collected for the DPPH assay. A 25 µL portion of the supernatant was combined with 975 µL of DPPH solution in an Eppendorf tube and incubated at room temperature for 30 min. After incubation, absorbance was measured at 520 nm, with methanol serving as the blank. The antioxidant activity was calculated using the following formula.

% DPPH radicals scavenging activity = Ab − As Ab × 100

In this formula, Ab represents the absorbance of the blank reading (methanol), while As denotes the absorbance of the sample.

2.6.2 Determination of total phenolic content (TPC)

For the estimation of total phenolic content in the soymilk samples, the Folin-Ciocalteu method was used with slight modifications (Zhao and Shah, 2014). 1 mL of soymilk sample was dissolved or mixed with methanol and centrifuged to obtain the methanol extract. A 10 µL methanol extract was combined with 790 µL of double-distilled water and 50 µL of Folin-Ciocalteu reagent (1N). Additionally, 150 µL of sodium carbonate was added to the mixture, which was then incubated at 40 °C in a water bath for 30 min. The absorbance was recorded at 765 nm.

2.6.3 Determination of phytic acid

For the estimation of phytic acid in the soymilk sample, an assay kit (Megazyme, K-PHYT, Wicklow, Ireland) (Tomar et al., 2021).

2.6.4 Determination of trypsin inhibitor unit

The method of measuring trypsin inhibitor was based on Ge et al. (2021) With minor modifications. 1 mL of fermented soymilk was dissolved in 8–10 mL of 0.01 N NaOH solution, adjusted to pH 8.4 to 10, and kept in a shaker for 3 h, then the aliquot (∼4 mL). The collected 4 mL samples were divided equally (2 mL in each test tube). Add 2 mL of trypsin solution (20 μg/mL) with 2 mL aliquot and keep at 37 °C in a water bath for 10 min afterward add 5 mL BAPNA solution (0.4 mg/mL) then stir the mixture and keep at 37 °C in a water bath for 10 min then further add 1 mL acetic acid for terminate the reaction and record the absorbance at 410 nm (A410S). For another 2 mL aliquot, 5 mL BAPNA solution (0.4 mg/mL) and 1 mL acetic acid were added to a test tube before adding trypsin solution (2 mL) as the reagent blank and recording the absorbance at 410 nm (A410SB).

Similarly, a 4 mL sample aliquot was replaced with distilled water, and the protocol was repeated to get the absorbance at 410 nm (A410R and A410RB). The following formula was used for calculating the trypsin inhibitor unit (TIU)-

T r y p s i n i n h i b i t i o n u n i t T I U m g s a m p l e = A 410 R − A 410 R B − A 410 S − A 410 S B × 100 × m l d i l u t e d s o y e x t r a c t m g s a m p l e m l d i l u t e d s o y e x t r a c t u s e d f o r t h e a s s a y

2.6.5 Soy isoflavone extraction and quantification

2.6.5.1 Sample preparation for isoflavone analysis in UHPLC-

Isoflavone analysis in the soymilk samples was done with ultra-high performance liquid chromatography (UHPLC) (Shimadzu CTO-20AC, Nexera XR, SPD-M20A, RID-20A, made in Japan) by using the method described by Lee et al. (2015) With slight modification. 1 mL of soymilk sample was mixed with 4 mL methanol (100%) and vortexed before being kept in incubation for 30 min at 70 °C. Centrifuge the tube with 13,000 rpm at 20 °C for 30 min. After centrifugation, 1 mL of the sample was filtered through a membrane filter (0.22 µm), and 4 µL of the sample was further analysed in UHPLC.

Soy isoflavones in the sample were quantified using UHPLC under the following conditions: a C18 silica column served as the nonpolar stationary phase, while a binary gradient of acetonitrile (10% acetonitrile as solvent A and 38% acetonitrile as solvent B) was used as the mobile phase. The mobile phase followed this time program (% solvent A/solvent B): 0 min (0/100), 5 min (10/90), 20 min (0/100), and 25 min (0/100). The flow rate was maintained at 0.8 mL/min for 25 min, with the column oven temperature set at 40 °C. Isoflavone resolution was monitored at 260 nm using a UV detector.

2.6.6 Raffinose family oligosaccharides analysis

2.6.6.1 Sample preparation for RFOs analysis in HPLC-

The raffinose family oligosaccharides (RFOs), including raffinose and stachyose, were analyzed following the method of Hati et al. (2014) with slight modifications. A 5 mL soymilk sample was dried in an oven at 60 °C until a constant weight was achieved, converting it into powder form. A 0.5 g portion of the dried sample was then dissolved in 5 mL of HPLC-grade water, shaken for 15 min, and centrifuged at 4000 g for 15 min. After centrifugation, 2.5 mL of the supernatant was mixed with 4 mL of HPLC-grade acetonitrile and incubated at room temperature for 2 h to precipitate soluble proteins. The mixture was then centrifuged at 3670 g for 5 min, and the supernatant was collected. A 0.5 mL aliquot of the supernatant was evaporated to dryness in a hot air oven, and the resulting residue was dissolved in 1 mL of 0.005 M H2SO4 prepared with HPLC-grade water. The solution was then filtered through a 0.2-micron syringe filter and transferred to HPLC vials for further analysis.

The RFO profiling of the prepared samples was conducted using an Agilent 1100 series High-Performance Liquid Chromatography (HPLC) system equipped with a stainless-steel Agilent Hi-Plex-H analytical column. The mobile phase consisted of 0.005 M H2SO4 in HPLC-grade water, prepared by dissolving 0.550 mL of H2SO4 in 2 L of HPLC water. The column temperature was maintained at 30 °C, with a flow rate of 0.8 mL/min and an injection volume of 20 µL for all samples. To determine the retention time of each component, a working sample of known concentration from a stock solution was analyzed separately. Peak identification was based on retention time and sample spiking for area calculation. Each sugar exhibited distinct retention times: raffinose at 8.44 min, stachyose at 8.02 min, and sucrose at 9.25 min. The concentrations of raffinose, stachyose, and sucrose were quantified using a standard regression curve generated from external standards obtained from Sigma-Aldrich.

2.7 Technological analysis of fermented soymilk

2.7.1 Color

The color analysis of the fermented soymilk sample was performed using a Hunter Lab colorimeter (LabScan XE Spectrophotometer by Hunter Lab, using the D65 illuminant with an angle of observation of 10°. 50 mL of soymilk samples were tempered to 20 °C before analysis. All three values L, a, and b*, were measured in triplicate in each sample (dos Santos et al., 2019).

2.7.2 Total soluble solids (TSS)-

TSS of the samples was determined using the handheld digital refractometer (IR 140) (Khubber et al., 2021).

2.7.3 Percentage of sedimentation

For the measurement of % sedimentation in the soymilk sample, the Saeidy et al. (2014) method with slight modification. Soymilk was accurately weighed (10 g) and poured into a falcon tube, then centrifuged at 4200 rpm at 25 °C for 5 min. After the centrifugation, discard the supernatant and place the falcon tube upside down for 5 s. Further wet sediments were transferred to a dry Petri plate (already weighed) and kept the Petri plate dry at 90 °C until a stable weight was obtained. % Sedimentation was calculated by using the following formula.

% S e d i m e n t a t i o n = B A × 100

Here A is the weight of the sample taken initially, and B is the weight of the sample after drying at 90 °C.

2.7.4 Rheological property

Rheological parameter studies were performed on the chilled samples after they had been equilibrated at 25 °C for 1 h. Using the cup and van geometry, rheology investigations of soymilk samples were conducted in a rheometer (Modular compact rheometer MCR52, with Anton Paar software) (Liu et al., 2004). 20 mL soymilk samples were poured into the cup and placed at the rheometer platform against the probe at 25 °C, and the curve with 1–100 measuring points. The rheological parameters such as Shear rate (γ, 1/s.), Shear stress (τ, Pa), Viscosity (η, Pa-s.), and flow index (n) were obtained through fore mentioned rheological analysis.

2.7.5 Sensory evaluation

Sensory evaluation was conducted after 48 h of fermentation at the Laboratory of Sensory Analysis, Division of Food Science and Post-Harvest Technology, ICAR–IARI, New Delhi. A panel of 20 untrained adult judges (70% aged 25–35 years and 30% aged 35–60 years) evaluated coded 40 mL soymilk samples served at 10 °C in individual sensory booths. Before evaluation, the panelists were briefed on the sensory attributes and the use of the 9-point hedonic scale (extreme dislike = 1, very much dislike = 2, moderate dislike = 3, mild dislike = 4, neither dislike nor like = 5, small like = 6, moderate like = 7, very much like = 8, and highly like = 9) and instructed to stir the samples before tasting. The samples were evaluated for colour, flavour, taste, texture, mouthfeel, and overall acceptability. Participation was voluntary, informed consent was obtained from all panelists, and the study was conducted in accordance with institutional ethical guidelines.

2.8 Statistical analysis

The experiment was conducted using a completely randomized design (CRD) with three independent biological replicates. Each biological replicate was analyzed in triplicate (technical replicates), and the mean value was used for statistical analysis. The experimental unit consisted of an independently prepared and fermented soymilk batch for each soybean variety. Data were subjected to two-way analysis of variance (ANOVA) to evaluate the effects of soybean variety, fermentation time, and their interaction. When significant differences were detected, mean comparisons were performed using Tukey’s honestly significant difference (HSD) test with Statistica 6.0 software (StatSoft Inc., Tulsa, OK, USA). Differences were considered statistically significant at p < 0.05. Results are presented as mean ± standard deviation (SD).

Principal component analysis (PCA) was performed using http://mvapp.kaust.edu.sa/MVApp to explore the multivariate relationships among the fermented soymilk samples. The analysis included 19 nutritional, functional, technological, and sensory variables selected to comprehensively characterize the effects of soybean variety and fermentation. Before analysis, all variables were standardized (z-score normalization) to eliminate differences in measurement scales and ensure equal contribution of each variable. PCA was used as an exploratory multivariate technique to visualize sample clustering and variable associations rather than to establish a definitive ranking of the soybean varieties.

3 Results and discussion

3.1 Changes in chemical compositions and viability of Lactobacillus acidophilus in fermented soymilk

The chemical compositions of protein, fat, crude fiber, carbohydrate, energy, total titratable acidity, and pH of all the investigated soybean varieties were determined, and the results are mentioned in Table 1. The protein content was increased after 48 h of fermentation in all three samples. A significantly higher protein content (p < 0.05), 4.70%, was observed in fermented black soymilk (BSM + LA). Whereas the fat content was reduced, opposite to protein content. The lowest fat content 1.45% was observed in the fermented food-grade soymilk (FGSM + LA). The crude fiber content was enhanced after the fermentation. A high amount of crude fiber (0.063%) was observed in the fermented black soymilk sample. The total carbohydrate content was increased in all the fermented soymilk samples. The highest carbohydrate content (5.04%) was found in the FGSM + LA sample. The energy of the soymilk samples was increased after 48 h of fermentation. The highest amount of energy (52.46 Kcal) was reported in the fermented soymilk sample.

TABLE 1. Changes in the chemical composition of different soymilks after 48 h of fermentation.

SampleFermentation time (h)Protein (%)Total fat (%)Crude fiber (%)Carbohydrate (%)Energy (Kcal)TTA (%)pH
FGSM + LA01.83 ± 0.076d1.93 ± 0.008c0.008c3.02 ± 0.012c36.77c1.23 ± 0.045d6.3 ± 0.11b
482.23 ± 0.076b1.45 ± 0.047f0.055b5.04 ± 0.073a42.15b1.50 ± 0.058c4.7 ± 0.11e
LKSM + LA01.51 ± 0.087e2.05 ± 0.081b0.005c2.30 ± 0.077e33.69d0.88 ± 0.009e6.7 ± 0.21a
482.14 ± 0.087c1.70 ± 0.041d0.045b4.57 ± 0.016b42.22b1.23 ± 0.014a4.5 ± 0.04f
BSM + LA01.11 ± 0.099f3.1 ± 0.078a0.009c2.46 ± 0.029d42.18b1.76 ± 0.043b6.9 ± 0.02a
484.70 ± 0.099a2.7 ± 0.051e0.063a2.32 ± 0.063e52.46a3.53 ± 0.156a3.9 ± 0.15g

Here, FGSM + LA, is Lactobacillus acidophilus fermented food-grade soymilk, LKSM + LA, is Lactobacillus acidophilus fermented low KTI, soymilk, and BSM + LA, is Lactobacillus acidophilus fermented black soymilk. All data represent the mean ± standard deviation of three independent experiments (n = 3), and different superscript letters indicate statistically significant differences (Tukey’s test, p < 0.05).

The viability cell counts in the log cfu/mL indicated that during the fermentation, the viability of L. acidophilus significantly (p < 0.05) increased in all three samples up to 48 h, as shown in Figure 2. The BSM shows high viability (9.6 log cfu/mL) in comparison to FGSM (8.8 log cfu/mL) and LKSM (8.4 log cfu/mL) samples. Similarly, the total titratable acidity (TTA) was also analyzed, and found that TTA values increased after 48 h fermentation in all three samples. The TTA value was high (3.53%) in the BSM + LA sample. Similarly, the pH of the samples was reduced during the fermentation, and a pH of 3.9 was the lowest pH observed in the BSM + LA sample at 48 h of fermentation. These results indicate L. acidophilus 1132 grow better in the black soymilk and produce acids responsible for the reduction in the TTA and pH of the sample. At a pH above 4.5, the flavor of the product is preserved due to the masking of volatile soybean compounds, such as n-hexanal and pentanal, by fermentation by-products like lactic acid, acetaldehyde, and diacetyl, which enhance the sensory quality of soymilk (Behrens et al., 2004; dos Santos et al., 2019). Therefore, maintaining a pH greater than 4.5 in fermented soymilk is desirable for optimal flavor. The changes in the viability count, TTA, and pH could be attributed to the availability of carbon sources such as carbohydrates, pigments, and other fatty acids (dos Santos et al., 2019). The black soybeans are rich in pigments like anthocyanins, which act as a prebiotic to enhance the probiotic count and reduce the TTA and pH of the fermented black soymilk (Sasi et al., 2023).

Viable cell counts of *Lactobacillus acidophilus* during fermentation of soymilk samples. Here, FGSM + LA is *Lactobacillus acidophilus* fermented food-grade soymilk, LKSM + LA is *Lactobacillus acidophilus* fermented low KTI soymilk, and BSM + LA is *Lactobacillus acidophilus* fermented black soymilk.
Figure 2. Viable cell counts of Lactobacillus acidophilus during fermentation of soymilk samples. Here, FGSM + LA is Lactobacillus acidophilus fermented food-grade soymilk, LKSM + LA is Lactobacillus acidophilus fermented low KTI soymilk, and BSM + LA is Lactobacillus acidophilus fermented black soymilk.

Iron (Fe), calcium (Ca), potassium (K), and zinc (Zn) were the minerals that contributed most to the Recommended Daily Intake. As shown in Table 2, all three soybean samples exhibited significant change (p < 0.05) in minerals (Fe, Zn, Ca, and K) content after fermentation. The iron (0.53 mg/100 g), zinc (0.51 mg/100 g), and calcium (17.28 mg/100 g) content were high in the BSM + LA sample after 48 h of fermentation. Fermentation significantly influenced the measured concentrations of minerals in the fermented soymilk samples, with BSM exhibiting comparatively higher levels of calcium, potassium, iron, and zinc than the other soybean varieties. However, it should be noted that fermentation in a closed system does not increase the absolute mineral content. The apparent increase in mineral concentration is more likely attributable to improved mineral extractability resulting from the degradation of mineral-binding antinutritional compounds, particularly phytic acid, as well as changes in dry matter composition and concentration effects during fermentation. Similar observations have been reported in fermented soybean products, where microbial phytase activity enhances mineral bioaccessibility by releasing minerals previously bound in phytate complexes, rather than generating new minerals (Egli et al., 2002; Frias et al., 2008). The observed increase in mineral concentration likely reflects enhanced extractability and bioaccessibility due to phytate degradation, together with changes in sample composition during fermentation, rather than de novo mineral synthesis. Iron deficiency is the most common cause of anemia globally. The treatment strategy encompasses prevention, food fortification (guaranteed by current legislation), and iron supplementation (Lopez et al., 2016). Iron is the most difficult micronutrient to add to fortified foods because iron salts can be quite unstable, and its oxidation depends on temperature and air exposure, which may produce adverse sensorial changes (Blanco-Rojo and Vaquero, 2019). The calcium content was high (49.99 mg/100 g) in the FGSM + LA sample.

TABLE 2. Mineral contents and ratio of different soymilk after 48 h of fermentation.

SampleFermentation time (h)Iron (mg/100 gZinc (mg/100 gCalcium (mg/100 g)Potassium (mg/100 g)
FGSM + LA00.33 ± 0.08g0.17 ± 0.02i7.84 ± 0.41h15.49 ± 0.26L
480.39 ± 0.03efg0.27 ± 0.02efg8.95 ± 0.82fgh49.99 ± 0.84a
LKSM + LA00.35 ± 0.02g0.24 ± 0.02gh9.29 ± 0.59efgh34.29 ± 0.56f
480.37 ± 0.02fg0.27 ± 0.04efg8.89 ± 0.62fgh45.34 ± 0.48b
BSM + LA00.36 ± 0.02fg0.20 ± 0.02hi8.17 ± 0.69h18.45 ± 0.77k
480.53 ± 0.03b0.51 ± 0.03a17.28 ± 0.81a39.96 ± 0.55d

Here, FGSM + LA, is Lactobacillus acidophilus fermented food-grade soymilk, LKSM + LA, is Lactobacillus acidophilus fermented low-KTI, soymilk, and BSM + LA, is Lactobacillus acidophilus fermented black soymilk. All data represent the mean ± standard deviation of three independent experiments (n = 3), and different superscript letters indicate statistically significant differences (Tukey’s test, p < 0.05).

3.2 Total phenolic content and antioxidant activity of fermented soymilk

As shown in Table 3, the total phenolic content (TPC) and antioxidant activity significantly (p < 0.05) increased after fermentation in all three soymilk samples. Phenolic compounds naturally found in soy milk are a group of phenolic acids and flavonoids. The TPC level in food-grade soymilk was increased from 0.66 ± 0.02 mgGAE/mL to 1.08 ± 0.02 mgGAE/mL after 48 h of fermentation. The TPC levels in low KTI soymilk and black soymilk were also increased from 1.07 ± 0.025 mgGAE/mL to 1.94 ± 0.003 mgGAE/mL and 0.7 ± 0.02 mgGAE/mL to 1.07 ± 0.02 mgGAE/mL after the 48 h fermentation, respectively. TPC values observed in the present study are comparable with a previous study that explored the impact of probiotic fermentation methods on TPC values of soymilk (1.72–3.20 mg GAE/g) (Xu and Chang, 2009b). The significant increase in total phenol content (p < 0.05) may be due to the enrichment of compounds with strong electron-donating properties, including intrinsic antioxidants, reducing peptides produced through bacterial fermentation, and aglycones isoflavone (AI), which aid in reducing the Fe-C reagent under alkaline conditions. In black soymilk, anthocyanin pigments may also contribute to variations in TPC levels during fermentation.

TABLE 3. Changes in functional parameters of different soymilks after 48 h of fermentation.

SampleAntioxidant activity (% DPPH scavenging)Total phenolic content (mg GAE/mLPhytic acid (g/100 gTrypsin inhibitor (TIU/mg sample
0 h48 h0 h48 h0 h48 h0 h48 h
FGSM + LA6.86 ± 0.103h13.26 ± 0.131a0.66 ± 0.02hi1.08 ± 0.02d1.1 ± 0.04a0.71 ± 0.04de101.84 ± 0.91a63.47 ± 1.09g
LKSM + LA1.22 ± 0.05k11.28 ± 0.05c1.07 ± 0.025d1.94 ± 0.003ef0.83 ± 0.042b0.65 ± 0.04f60.12 ± 1.25g38.37 ± 1.56k
BSM + LA3.9 ± 0.32j6.55 ± 0.15h0.7 ± 0.02h1.07 ± 0.02d0.12 ± 0.004jk0.10 ± 0.03j96.63 ± 2.18b70.58 ± 1.65ef

Here, FGSM + LA, is Lactobacillus acidophilus fermented food-grade soymilk, LKSM + LA, is Lactobacillus acidophilus fermented low KTI, soymilk, and BSM + LA, is Lactobacillus acidophilus fermented black soymilk. All data represent the mean ± standard deviation of three independent experiments (n = 3), and different superscript letters indicate statistically significant differences (Tukey’s test, p < 0.05).

Similarly, fermentation significantly enhanced the in vitro antioxidant capacity of all fermented soymilk samples, as evidenced by the increased DPPH radical-scavenging activity (Table 3). The DPPH radical scavenging ability of the soymilk samples was also significantly (p < 0.05) increased in all three samples after 48 h fermentation. The antioxidant activity of the food grade soymilk increased from 6.86 ± 0.103 μmol TE/g to 13.26 ± 0.131 μmol TE/g. The low KTI soymilk and black soymilk also exhibited enhanced antioxidant activity from 1.22 ± 0.05 μmol TE/g to 11.28 ± 0.05 μmol TE/g and 3.9 ± 0.32 μmol TE/g to 6.55 ± 0.15 μmol TE/g, respectively. Concerning the various chemical structures of individual antioxidants and HPLC analysis data, some hydrophilic compounds such as vitamins B and C, phenolic acids, and isoflavones were expected to contribute to antioxidant activity in aqueous extracts, whereas vitamin E contributed to lipophilic antioxidant capacity (Rodríguez-Roque et al., 2013). This improvement is likely associated with the increased release of phenolic compounds and the bioconversion of isoflavone glucosides into their corresponding aglycone forms during fermentation. The enhanced in vitro antioxidant capacity indicates an improved ability of the fermented soymilk extracts to scavenge free radicals under experimental conditions. However, these findings should not be interpreted as direct evidence of physiological or clinical antioxidant effects in humans, as the bioavailability, metabolism, and in vivo efficacy of these bioactive compounds require further investigation through animal and human studies. The presence of phenolics and other antioxidant compounds like anthocyanins helps to achieve high antioxidant activity in the fermented black soymilk sample.

3.3 Changes in antinutritional factors and raffinose family oligosaccharide (RFOs)

Off-flavors and antinutritional factors (ANFs) pose significant challenges to the consumer acceptance of soybeans as a food source. As presented in Table 3, antinutritional factors like phytic acid and trypsin inhibitor activity showed significant reduction (p < 0.05) after fermentation in all three soymilk samples. Phytic acid is the metal chelating ANF. For the food-grade soymilk, the phytic acid content reduced from 1.1 ± 0.04 g/100 g to 0.71 ± 0.04 g/100 g, for the low KTI soymilk sample, phytic acid reduced from 0.83 ± 0.042 g/100 g to 0.65 ± 0.04 g/100 g and for the black soymilk sample, phytic acid reduced from 0.12 ± 0.004 g/100 g to 0.10 ± 0.03 g/100 g after the 48 h of the fermentation. A similar trend in the reduction of the phytic acid was found in the fermented soymilk reported in other research findings (García-Mantrana et al., 2015).

Similarly, the trypsin inhibitor activity reduced from 101.84 ± 0.91 TIU/mg to 63.47 ± 0.91 TIU/mg in the FGSM, from 60.12 ± 1.25 TIU/mg to 38.37 ± 1.56 TIU/mg in the LKSM, and from 96.63 ± 2.18 TIU/mg to 70.58 ± 1.65 TIU/mg in the black soymilk samples after 48 h of fermentation. The present study aligns with the previous study where the trypsin inhibitor was minimized from 1.21 to 0.011 mg/g (99%) in fermenting soybeans by Lactobacillus plantarum (Adeyemo and Onilude, 2013). Although fermentation further reduced trypsin inhibitor activity and phytic acid, the observed decreases should be interpreted as the combined effects of soaking, dehulling, thermal processing, autoclaving, and microbial fermentation. In particular, thermal treatment likely inactivated a substantial proportion of heat-labile trypsin inhibitors before fermentation, whereas microbial phytase and other enzymatic activities contributed to additional degradation during fermentation (Abbas and Ahmad, 2018).

Soybean contains flatus-producing non-hydrolyzable carbohydrates like the raffinose family oligosaccharides (RFOs). Flatulence in humans is due to the lack of the enzyme α-galactosidase in the gut, a foremost requisite for the hydrolysis of the α-galactoside bonds of raffinose and stachyose for synthesizing readily digestible sugars (Shabalin et al., 2002). Raffinose family oligosaccharides (RFOs) is determined through chromatography, and the results are shown in Table 4. The characteristic HPLC chromatogram peaks of raffinose, stachyose, and sucrose are shown in Figure 3. The RFOs (raffinose and stachyose) content was significantly (p < 0.05) reduced during the L. acidophilus-employed fermentation. In food-grade soymilk, the raffinose reduced from 8.7 ± 0.09 mg/L to 6.81 ± 0.14 mg/L, and stachyose reduced from 25.56 ± 1.04 mg/L to 20.33 ± 0.25 mg/L. In low KTI soymilk, raffinose reduced from 5.8 ± 0.16 mg/L to 3.08 ± 0.14 mg/L, and stachyose reduced from 20.11 ± 0.25 mg/L to 15.98 ± 0.24 mg/L. In the black soymilk, raffinose reduced from 2.5 ± 0.89 mg/L to 0.89 ± 0.08 mg/L, and stachyose reduced from 15.98 ± 0.68 mg/L to 2.19 ± 0.73 mg/L. The sucrose content also declined during the fermentation. Sucrose content reduced from 40.12 ± 1.54 mg/L to 14.65 ± 1.1 mg/L, 36.94 ± 0.84 mg/L to 10.28 ± 0.20 mg/L, and 15.98 ± 0.68 mg/L to 2.19 ± 0.73 mg/L during 48 h fermentation of the FGSM, LKSM, and BSM, respectively. It has been proposed that the predominant α-galactosyl oligosaccharides in soymilk, such as raffinose and stachyose, serve as an effective fermentable substrate for the growth of L. acidophilus. Furthermore, the levels of stachyose and raffinose in soymilk were reduced after 48 h of fermentation with L. acidophilus. Similar results are also reported in various studies (Pinthong et al., 1980; Wang et al., 1974; Scalabrini et al., 1998; Pyo et al., 2005). In the fermented black soymilk sample, the reduction of the oligosaccharides was high, which might be due to fulfilling the requirement of the probiotic to grow and proliferate.

TABLE 4. Changes in oligosaccharides (raffinose and stachyose) content in different soymilks after 48 h of fermentation.

SampleFermentation time (h)Raffinose (mg/L)Stachyose (mg/L)Sucrose (mg/L)
FGSM + LA08.7 ± 0.09a25.56 ± 1.04a40.12 ± 1.54a
486.81 ± 0.14b20.33 ± 0.25b14.65 ± 1.1e
LKSM + LA05.80 ± 0.16d20.11 ± 0.25b36.94 ± 0.84b
483.08 ± 0.14g15.98 ± 0.24d10.28 ± 0.20g
BSM + LA02.50 ± 0.06h15.13 ± 0.30d15.98 ± 0.68d
480.89 ± 0.08i1.13 ± 0.04j2.19 ± 0.73L

Here, FGSM + LA, is Lactobacillus acidophilus fermented food-grade soymilk fermented, LKSM + LA, is Lactobacillus acidophilus fermented low KTI, soymilk, and BSM + LA, is Lactobacillus acidophilus fermented black soymilk. All data represent the mean ± standard deviation of three independent experiments (n = 3), and different superscript letters indicate statistically significant differences (Tukey’s test, p < 0.05).

Raffinose family Oligosaccharides (RFOs) profiling of fermented and non-fermented soymilk samples. Here, FGSM + LA is *Lactobacillus acidophilus* fermented food-grade soymilk, LKSM + LA is *Lactobacillus acidophilus* fermented low KTI soymilk, and BSM + LA is *Lactobacillus acidophilus* fermented black soymilk.
Figure 3. Raffinose family Oligosaccharides (RFOs) profiling of fermented and non-fermented soymilk samples. Here, FGSM + LA is Lactobacillus acidophilus fermented food-grade soymilk, LKSM + LA is Lactobacillus acidophilus fermented low KTI soymilk, and BSM + LA is Lactobacillus acidophilus fermented black soymilk.

3.4 Isoflavones bioconversion during fermentation

Lactic acid bacteria (LAB) and bifidobacteria play a crucial role in deglycosylating isoflavone conjugates during the fermentation of soy foods. Table 5 presents the changes in isoflavone concentrations during the fermentation of all three soymilk varieties. The characteristic HPLC chromatograms of the isoflavones detected in the L. acidophilus fermentation of the three soybean varieties are shown in Figure 4. The results showed a decrease in the first three peaks (daidzin, glycitin, genistin) of the glucoside isoflavones (GI) and an increase in the later three (daidzein, glycitein, genistein) peaks of the aglycone isoflavones (AI).Previous studies have also mentioned soymilk as a good source of isoflavones in their glucoside, aglycone, acetylated, and malonylated forms (Yu et al., 2021).

TABLE 5. Changes in isoflavone concentration (mg/100 g) in different types of soymilks after 48 h of fermentation.

SamplesFGSM + LALKSM + LABSM + LA
Type of isoflavone0 h48 h0 h48 h0 h48 h
Diadzin3.53 ± 0.15e9.02 ± 0.31d40.79 ± 0.47a9.97 ± 0.26c11.83 ± 0.01b10.26 ± 0.22c
Glycitin4.79 ± 0.06c3.02 ± 0.07d10.18 ± 0.37a3.4 ± 0.12d6.12 ± 0.25b1.24 ± 0.04e
Genistin6.71 ± 0.176c4.71 ± 0.04d8.2 ± 0.14b0.86 ± 0.02f7.53 ± 0.26a2.93 ± 0.07e
Total glucoside isoflavones (GI)15.042.2039.170.0 ± 0.025.492.94
Diadzein2.26 ± 0.006e34.93 ± 0.53a13.89 ± 0.05c20.48 ± 0.53b8.35 ± 0.128d19.63 ± 0.159b
Glycitein1.4 ± 0.04f8.52 ± 0.22c12.33 ± 0.17b31.99 ± 0.14a7.47 ± 0.16d5.67 ± 0.08e
Genistein0.00 ± 0.00f5.91 ± 0.26a4.58 ± 0.09c5.16 ± 0.07b1.89 ± 0.01e3.71 ± 0.14d
Total aglycone isoflavones (AI)1.4449.370.0 ± 0.057.640.0 ± 0.029.03
Total isoflavones16.4851.5739.1757.6425.4931.97

Here, FGSM + LA, is Lactobacillus acidophilus fermented food-grade soymilk, LKSM + LA, is Lactobacillus acidophilus fermented low KTI, soymilk, and BSM + LA, is Lactobacillus acidophilus fermented black soymilk. All data represent the mean ± standard deviation of three independent experiments (n = 3), and different superscript letters indicate statistically significant differences (Tukey’s test, p < 0.05).

Isoflavone profiling in fermented and non-fermented soymilk samples. Here, FGSM + LA is *Lactobacillus acidophilus* fermented food-grade soymilk, LKSM + LA is *Lactobacillus acidophilus* fermented low KTI soymilk, and BSM + LA is *Lactobacillus acidophilus* fermented black soymilk.
Figure 4. Isoflavone profiling in fermented and non-fermented soymilk samples. Here, FGSM + LA is Lactobacillus acidophilus fermented food-grade soymilk, LKSM + LA is Lactobacillus acidophilus fermented low KTI soymilk, and BSM + LA is Lactobacillus acidophilus fermented black soymilk.

Fermentation resulted in a significant decrease in the glucoside isoflavones daidzin, glycitin, and genistin, accompanied by a corresponding increase in their respective aglycone forms, daidzein, glycitein, and genistein (Table 5). For example, daidzin decreased from 9.02 mg/100 g to 3.53 mg/100 g, while daidzein increased from 2.26 mg/100 g to 34.93 mg/100 g, indicating extensive bioconversion during fermentation. Similar trends were observed for the glycitin–glycitein and genistin–genistein pairs across all soybean varieties, with the greatest conversion observed in LKSM. The observed decrease in glucoside isoflavones together with the corresponding increase in aglycone isoflavones is attributed to the β-glucosidase activity of L. acidophilus. This enzyme hydrolyzes the β-glycosidic bond linking the glucose moiety to the isoflavone backbone, converting daidzin, glycitin, and genistin into their biologically active aglycone forms, namely, daidzein, glycitein, and genistein. Aglycone isoflavones exhibit greater intestinal absorption and bioavailability than their glucoside counterparts because they can be absorbed directly without prior hydrolysis in the gastrointestinal tract. Therefore, the enhanced aglycone content observed after fermentation reflects improved nutritional quality rather than an increase in total isoflavone synthesis (Langa et al., 2023).

The total isoflavone contents were the sum of total genistein, total daidzein, and total glycitein. The total isoflavone (TI) content was enhanced in fermented soymilk samples of all three varieties. In the food-grade soymilk, the TI increased from 16.48 mg/100 g to 51.57 mg/100 g, for low KTI soymilk, TI increased from 39.17 mg/100 g to 57.64 mg/100 g, and for the black soymilk, it increased from 25.49 mg/100 g to 31.97 mg/100 g. The decrease in bioconversion rate was possibly due to denaturation of β-glycosidase at lower pH (pH 4.0-FGSM, pH 4.2- LKSM, and pH 3.9- BSM after fermentation) (Martini et al., 1987). It is also reported that they exhibited higher glucosidase activity at low pH (3.85) (Zhao and Shah, 2014).

3.5 Changes in color, TSS, and % sedimentation during fermentation

As shown in Table 6, the L ***, a, and b<sup></sup> values of the color parameter significantly (p < 0.05) changed in all three samples after 48 h fermentation. The L *** value of the FGSM (80.22 ± 0.38) and LKSM (81.01 ± 0.21) was high in comparison to BSM (57.3 ± 0.29), which improved after fermentation. The color of fermented soymilk depends on pigments in the raw soybeans, which can vary by soybean variety.

TABLE 6. Changes in color, TSS, and %sedimentation in different soymilks after 48 h of fermentation.

SampleColorTSS (% brix)% sedimentation
L ***a ***b ***
0 h48 h0 h48 h0 h48 h0 h48 h0 h48 h
FGSM + LA80.22 ± 0.38cd78.37 ± 0.52h0.02 ± 0.001n1.05 ± 0.01ij14.22 ± 0.22L15.88 ± 0.26d6.0 ± 0.03b2.6 ± 0.06g4.2 ± 0.01j7.5 ± 0.27e
LKSM + LA81.01 ± 0.21b77.43 ± 0.12j1.87 ± 0.12f1.68 ± 0.03g16.06 ± 0.05a15.7 ± 0.34f5.0 ± 0.13c3.8 ± 0.07d3.6 ± 0.09k6.7 ± 0.04f
BSM + LA57.3 ± 0.29n60.11 ± 0.71m4.6 ± 0.15d5.57 ± 0.04b6.23 ± 0.17q7.98 ± 0.31o7.0 ± 0.11a3.1 ± 0.06f5.1 ± 0.11i11.77 ± 0.08a

Here, FGSM + LA, is Lactobacillus acidophilus fermented food-grade soymilk, LKSM + LA, is Lactobacillus acidophilus fermented low KTI, soymilk, and BSM + LA, is Lactobacillus acidophilus fermented black soymilk. L *** represents the lightness, with values from 0 (black) to 100 (white), a* positive values are red and negative values are green, b* positive values are yellow and negative ones are blue. All data represent the mean ± standard deviation of three independent experiments (n = 3), and different superscript letters indicate statistically significant differences (Tukey’s test, p < 0.05).

Total soluble solids (TSS) of all three samples significantly (p < 0.05) reduced during the fermentation, as displayed in Table 6. For FGSM, TSS reduced from 6.0% ± 0.03% brix to 2.6% ± 0.06% brix, for LKSM TSS reduced from 5.0% ± 0.13% brix to 3.8% ± 0.07% brix, and for BSM TSS reduced from 7.0% ± 0.11% brix to 3.1% ± 0.06% brix at 48 h of fermentation. Present findings aligned with the reported TSS of the fermented soymilk sample (5.26% Brix) (dos Santos et al., 2019). According to Kristo et al. (2003), a lower total solids count indicates a lower buffering capacity, which in turn indicates a greater decrease in pH for the same amount of acid produced.

Physical stability is an important parameter from a sensorial point of view (Granato et al., 2012), and a change in pH may cause the protein in beverages to precipitate, thus increasing sedimentation. The higher the sedimentation, the lower the stability of the product, as a consequence of particle sedimentation (Cruz et al., 2007). The sedimentation of all three soymilk samples significantly (p < 0.05) increased after fermentation, as shown in Table 6. For FGSM, sedimentation increased from 4.2% ± 0.01% to 7.5% ± 0.01%, for LKSM sedimentation increased from 3.6% ± 0.09% to 6.7% ± 0.04%, and for BSM sedimentation increased from 5.1% ± 0.11% to 11.77% ± 0.08% after 48 h fermentation. The values found in this study were higher than those reported for soymilk (Cruz et al., 2007; Poliseli-Scopel et al., 2012), showing that soymilk has lower stability after fermentation. The particles of the black soymilk dispersed in the aqueous phase have varying characteristics and native protein aggregates (protein bodies), polymerization of the pigments and other aggregates might be responsible for the higher sedimentation in the sample.

3.6 Viscosity and sensory characteristics of the fermented soymilk

Figure 5 shows the changes in the viscosity in the fermented soymilk sample in all three varieties. The viscosity of all three fermented soymilk samples increased with decreasing the shear rate. The highest viscosity (19.0 Pa.S) was observed with a minimal shear rate (1.0 1/s) in the fermented black soymilk samples in comparison to the viscosity of the fermented food-grade soymilk (17.1 Pa.S) and fermented low KTI soymilk (13.7 Pa.S) samples with the same shear rate (1.0 1/s). The interactions between protein and polysaccharide affect the viscosity of the soymilk and during the probiotic fermentation, Lactobacillus species secretes the exopolysaccharides (EPS), also increasing the viscosity of the soymilk (Saeidy et al., 2014).

Viscosity of the soymilk samples. Here, FGSM + LA is *Lactobacillus acidophilus* fermented food-grade soymilk, LKSM + LA is *Lactobacillus acidophilus* fermented low KTI soymilk, and BSM + LA is *Lactobacillus acidophilus* fermented black soymilk.
Figure 5. Viscosity of the soymilk samples. Here, FGSM + LA is Lactobacillus acidophilus fermented food-grade soymilk, LKSM + LA is Lactobacillus acidophilus fermented low KTI soymilk, and BSM + LA is Lactobacillus acidophilus fermented black soymilk.

The sensory characteristics are also an important parameter for acquiring consumer acceptance besides nutritional and health-promoting features. The sensory parameters, including color, mouthfeel, consistency, taste, and flavor, of the soymilk samples were evaluated, and their overall acceptability based on these parameters is presented in Figure 6. The fermented soymilk samples of all three varieties showed better overall acceptability in comparison to nonfermented soymilk samples at 48 h of fermentation. Food-grade soybean variety exhibited better acceptability (6.19 ± 0.26) among the three varieties. However, the overall acceptability declined in all three samples after 28 days of storage at 4 °C, as shown in Figure 6.

Overall acceptability based on sensory evaluation (scale 1–9) of the fermented soymilk samples at 48 h and 28 days of storage. Here; FGSM + LA is *Lactobacillus acidophilus* fermented food-grade soymilk fermented, LKSM + LA is *Lactobacillus acidophilus* fermented low KTI soymilk, and BSM + LA is *Lactobacillus acidophilus* fermented black soymilk.
Figure 6. Overall acceptability based on sensory evaluation (scale 1–9) of the fermented soymilk samples at 48 h and 28 days of storage. Here; FGSM + LA is Lactobacillus acidophilus fermented food-grade soymilk fermented, LKSM + LA is Lactobacillus acidophilus fermented low KTI soymilk, and BSM + LA is Lactobacillus acidophilus fermented black soymilk.

A gradual decline in sensory acceptability was observed during refrigerated (4 °C) storage, with the lowest overall acceptability recorded after 28 days. This reduction is likely associated with continued post-fermentation metabolic activity of L. acidophilus, resulting in progressive acidification and the accumulation of organic acids that impart a sour flavour. In addition, storage-related physicochemical changes, including protein aggregation, slight phase separation, and changes in viscosity, may have negatively influenced mouthfeel and appearance. Although microbial viability was not monitored during storage in the present study, changes in the physiological state of the bacterial cells could also have influenced flavour development and overall sensory quality. These factors collectively contributed to the decline in consumer acceptability during prolonged refrigerated storage. Only sensory acceptability was evaluated during refrigerated storage, while microbial viability, pH, titratable acidity, and viscosity were not monitored. Therefore, the decline in sensory scores should be interpreted solely based on sensory evaluation. Future studies should include these parameters to comprehensively assess the storage stability and shelf life of fermented soymilk.

3.7 Correlation study and principal component analysis (PCA)

Principal component analysis (PCA) was performed on the nineteen variables, as shown in Figure 7. The first principal component (PC1) explains 50.7% of the total variance, while the second principal component (PC2) accounts for 40.2% (Figure 7A). Therefore, by utilizing just the first two principal components, PC1 and PC2, we can capture 90.9% of the total variance, providing significant insights into the soybean varieties. PCA revealed clear separation among the three fermented soymilk samples based on the combined variation of the 19 measured variables (Figure 7). The clustering pattern indicates differences in nutritional, functional, technological, and sensory characteristics among the soybean varieties. The proximity of variables and sample scores in the biplot illustrates their associations; however, PCA was used as an exploratory tool to visualize multivariate relationships and identify the major sources of variation rather than to establish a definitive ranking or superiority of any soybean variety.

Statistical analysis, **(A)** principal component analysis (PCA), and **(B)** correlation studies of nineteen variables.
Figure 7. Statistical analysis, (A) principal component analysis (PCA), and (B) correlation studies of nineteen variables.

In this study, fermented BSM showed high values of the desired traits like protein content, minerals (Fe, Ca, Zn, K) content, and a high value of titratable acidity with a low pH value, and high sedimentation % with a low TSS value. RFOs showed a rapid decline in fermented BSM compared to FGSM and LKSM. In fermented FGSM, we observed high antioxidant activity, along with low levels of phytic acid and trypsin inhibitor units. In fermented LKSM, the total phenolic content (TPC) was higher compared to both FGSM and BSM.

In the current study, the VL-Bhatt soybean variety demonstrated superior nutritional values, while the Karune variety exhibited greater reductions in anti-nutritional factors. The high concentration of pigmented molecules, such as anthocyanins, in black soybeans supports probiotic proliferation during soymilk fermentation by serving as an energy source (Kumar et al., 2023; Sasi et al., 2023; Hasan et al., 2024). Therefore, blending these two varieties could be an effective strategy for producing nutritionally and functionally enriched fermented soymilk. Alternatively, extracting anthocyanins from the seed coat and incorporating them into soymilk could also enhance its fermentation properties.

4 Conclusion

This study demonstrates that laboratory-scale fermentation of soymilk prepared from three soybean varieties using L. acidophilus under controlled conditions significantly influenced their nutritional, functional, technological, and sensory characteristics. Fermented black soymilk (BSM) exhibited higher protein and mineral (Fe, Ca, K, and Zn) contents, greater energy value, and lower raffinose family oligosaccharide (RFO) levels. In contrast, fermented food-grade soymilk (FGSM) showed lower concentrations of antinutritional factors and achieved the highest consumer acceptability, whereas fermented low-KTI soymilk (LKSM) demonstrated greater isoflavone bioconversion and higher total phenolic content. These findings indicate that soybean genotype plays an important role in determining the quality attributes of fermented soymilk. However, as the study was conducted under controlled laboratory conditions, further investigations at pilot and industrial scales are required to validate these results, optimize varietal blending strategies, and evaluate product stability and consumer acceptance during storage before commercial application.

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Footnotes

Edited by: Digambar Kavitake, Pondicherry University, India

Reviewed by: Sushmita Dwivedi, Govind Ballabh Pant Institute of Engineering and Technology, India

Hooman Shirvani, University of Ilam, Iran

Statements

Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The author MH declared that they were an editorial board member of Frontiers at the time of submission. This had no impact on the peer review process and the final decision.

Funding

The author(s) declared that financial support was not received for this work and/or its publication.

Data availability

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Author contributions

MH: Formal Analysis, Software, Writing – original draft, Writing – review and editing. DA: Data curation, Formal Analysis, Writing – original draft, Writing – review and editing. VK: Resources, Supervision, Writing – original draft, Writing – review and editing. AS: Project administration, Resources, Supervision, Writing – original draft, Writing – review and editing. AD: Conceptualization, Project administration, Resources, Supervision, Writing – original draft, Writing – review and editing.

Acknowledgements

The ICAR-Indian Agricultural Research Institute, New Delhi, Government of India, is thankfully acknowledged for providing the facility to conduct this research.

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Citation

Hasan, M., et al. (2026). Soybean variety influences the quality and functionality of soymilk fermented by Lactobacillus acidophilus 1132. Frontiers in Food Science and Technology, Volume 6. https://doi.org/
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Copyright © 2026 Hasan, Ahmad, Krishnan, Sachdev and Dahuja.

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*Correspondence: Muzaffar Hasan; Anil Dahuja

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